Metabolic imaging methods for assessment of oocytes and embryos

ABSTRACT

The invention provides novel non-invasive in vitro to methods for assessing the metabolic condition of oocytes and/or embryos with fluorescence lifetime imaging microscope, that can be used, for example, in assessment of oocytes and embryos in assisted reproductive technologies.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/750,061 filed Jan. 8, 2013, the contentof which are incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods for assessing the metabolic conditionof oocytes and/or embryos, and can be used, e.g., in assessment ofoocytes and embryos in assisted reproductive technologies.

BACKGROUND

Assisted Reproductive Technology (ART) has revolutionized the treatmentsof human infertility in the past 30 years and has become ubiquitous. ARTcurrently accounts for over 1% of birth in the U.S. (SART, 2005).However, success rates for ART are low, only 10%-35% of cycles result insuccessful birth, leading to high costs and use of multiple embryos,which in turn gives rise to high rates of multiple gestations. Multiplegestations greatly increase mortality rates and suffering for bothinfant and mothers, and produce substantial financial costs. It has beenestimated that complications from multiple pregnancy from ART accountfor approximately one billion dollars of health care cost annually inthe U.S. (Bromer and Seli, 2008).

One of the major reasons for the low success rate of ART is the absenceof reliable methods for selecting the highest quality embryo(s) fortransfer. The lack of methods for assessing embryo quality has led tosubstantial efforts to develop improved assays of embryo viability. Thecurrent most reliable method for predicting embryo quality is to examineembryo morphology prior to transfer using standard transmitted lightmicroscopy systems, with some clinics exploring the utility oftime-lapsing imaging using specialized microscopes such as theEmbryoScope by Unisense or EPIC by Auxogyn. However, selection criteriagenerally remain subjective and only result in the ˜35% success ratequoted above. Newly proposed non-microscopy based methods, usinggenomic, transcriptomic or proteomic based assays (Uyar, et al, 2012),require a biopsy of the embryo and are thus invasive and significantlyreduce rates of embryo survival (Scott et al., 2006). A metabolomicapproach which initially showed promise was to assay the metabolic stateof the embryo by measuring changes in metabolites in the embryo culturemedia (Botros, et al. 2008). However, a prospective randomized trial hasrecently failed to show that utilization of such metabolomic assessmentimproves selection over morphologic evaluation alone (Vergouw et al,2012).

Minimally invasive, simple and reliable methods for assessing embryo oroocyte viability for assisted reproductive technologies would provide asignificant advance for improving the safety of the mother and thefetus, and would also reduce the costs of the assisted reproductivetechnologies by reducing the number of times one has to try implantationto get pregnant and also by reducing multiple gestations.

SUMMARY OF THE INVENTION

We provide an integrated, automated system for rapidly determiningoocyte and embryo quality. The described methods are minimally invasive.We have discovered that a direct analysis of the metabolic state ofthese cells without additional manipulations of the cells or the cellculture media can be performed reliably and rapidly, and in most casesminimally invasively using fluorescence lifetime imaging microscopy(FLIM) of NADH and/or FAD in an in vitro assay.

Thus, we provide methods for assessing oocytes and embryos either usingoocytes or complete embryos or by using granulosa or cumulus cellsassociated with the oocytes or one or more cells biopsied from theembryo by analyzing their metabolic state using autofluorescence fromnicotinamide adenine dinucleotide (NADH) and/or flavin adeninedinucleotide (FAD) with FLIM. Cells with objectively measured adequatemetabolic state as indicated by fluorescent lifetime of protein boundand/or free NAHD or protein bound and/or free FAD indicate an oocyte orembryo that can be selected for in vitro fertilization or implantation,and if the metabolic state is inadequate the oocyte/embryo can bediscarded from in vitro fertilization or implantation.

For example, we showed that unperturbed oocytes exhibit a range ofvalues of alpha and beta (FIG. 1, circles). As seen in FIG. 1, oocytequality depends on oocyte metabolic state. Oocyte metabolic state can berapidly, non-invasively, and quantitatively measured by FluorescenceLifetime Imaging Microscopy (FLIM) of NAD or NADH. Two parameters (alphaand beta) are extracted from FLIM measurements of NADH (or NAD) inoocytes. In the example, we used mouse oocytes but similar calculationsare expected to work for human oocytes, as the mammalian cells, such asmouse and human cells are relatively similar, particularly relating totheir metabolic state, at this stage. Each point corresponds to datafrom a single oocyte. Perturbing oocytes by specific metabolicinhibitors (black crosses), or non-specific damage (triangles) causesboth parameters to increase. Unperturbed oocytes (circles) exhibit arange of values of alpha and beta. These parameters are indicative ofoocyte quality.

One can compare the NADH/FAD autofluorescence lifetime to a referencedistribution which provides a convenient way of selecting viable andnon-viable oocytes/embryos.

The lifetime distribution of NADH/FAD can be approximated as a sum oftwo exponentials. The parameter α is the ratio of the amplitude of thetwo exponentials, the parameter, β, is the lifetime of the longerexponential. Increase in both alpha and beta of NADH was found to beindicative of damage in the embryo/oocyte. Thus, typically, cells withincreased alpha and beta values compared to a reference value arediscarded from ART methods as they would be considered damaged and alphaand beta less than a reference value would be selected for ART methodsas their metabolic activity is indicative of healthy activity.

The FLIM measurements according to the methods of the invention can beperformed with extremely low levels of illumination, which is far lessthan is currently used in in vitro fertilization clinics to determinethe morphology of oocytes and embryos. Therefore, the FLIM measurementsperformed according to the methods of the present invention will notperturb oocytes and embryos and are thus as non-invasive as possible.

Accordingly, we provide a method for assessing the quality of an oocyteor an embryo, the method comprising (a) exposing a test cell selectedfrom the oocyte or an oocyte-associated cumulus cell or the embryo or acell from the embryo to a fluorescence lifetime imaging microscope(FLIM) to acquire a fluorescence lifetime histogram of auto-fluorescenceof endogenous NADH or FAD for the test cell; (b) averaging thefluorescence lifetime histogram of NADH auto-fluorescence or FADauto-fluorescence or both over the entire test cell, or the cytoplasmthe test cell, or mitochondria of the test cell; (c) comparing theaveraged fluorescence lifetime histogram from the test cell to anaveraged fluorescence lifetime histogram reference value to determine ifthe measured averaged fluorescence lifetime histogram from the test celldiffers statistically from that of the reference value; and (d) if theaveraged fluorescence lifetime histogram from the test cell does notdiffer statistically from the reference value then selecting the oocytefor in vitro fertilization or embryo for implantation and if theaveraged fluorescence lifetime histogram from the test cell differsstatistically from the reference value then excluding the oocyte from invitro fertilization or embryo from implantation.

In some aspects of all the embodiments of the invention, the methodcomprises a step of establishing the statistical significance by fittinglifetime histograms to a sum of two exponentials and the parameters ofthe measured cells are deemed to fall within or not fall within therange of parameters found in the healthy cells.

In some aspects of all the embodiments of the invention, the parametersto be compared between the measurements and the healthy cells are alpha,defined as the ratio of the amplitude of the two exponentials, and beta,defined as the lifetime of the longer exponential.

In some aspects of all the embodiments of the invention, the maximumvalue of alpha from the healthy cells is a specific value within therange 1.0-4.0 and the corresponding maximum value of beta of healthycells is in the range 2000 ps-3000 ps.

In some aspects of all the embodiments of the invention, the FLIM isperformed using a wavelength of about 740 nm in two-photon fluorescenceexcitation and using an emission bandpass filtered centered around about460 nm.

In some aspects of all the embodiments of the invention, the FLIM isperformed using a wavelength of about 340 nm in one-photon fluorescenceexcitation and using an emission bandpass filtered centered around about460 nm.

In some aspects of all the embodiments of the invention, the FLIM isperformed in the frequency domain instead of the time domain.

In some aspects of all the embodiments of the invention, the FLIM isperformed using a wavelength of about 900 nm in two-photon fluorescenceexcitation and using an emission bandpass filtered centered around about550 nm.

In some aspects of all the embodiments of the invention, the FLIM isperformed using a wavelength of about 450 nm in one-photon fluorescenceexcitation and using an emission bandpass filtered centered around about550 nm.

In all aspects of the invention, the methods are performed in vitro. Thenoninvasive nature of the methods allow them to be performed withoutharming the embryos or oocytes.

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent with reference to the detaileddescription of the preferred embodiments and to the accompanyingdrawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows data from FLIM imaging of NADH in mouse oocytes. Non-linearmicroscopy was used to assess the quality of live oocytes and embryos.FLIM curves from individual oocytes were fit to the sum of twoexponentials. The parameter α, is the ratio of the amplitude of the twoexponentials, the parameter, β, is the lifetime of the longerexponential in picoseconds. Each point corresponds to data from a singleoocyte. Unperturbed oocytes exhibit a range of values of α and β.Perturbing oocytes by specific metabolic inhibitors (circles) ornon-specific damage (triangles) causes both parameters to increase.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that the metabolic state of an oocyte or an embryocan be directly assessed using FLIM imaging of the autofluorescence ofthe cellular metabolites inside the oocyte or the embryo or using one ormore cumulus cells that surround the oocyte or a cell biopsied from theembryo. Oocytes and embryos with acceptable metabolic status can then beselected for in vitro fertilization or implantation and oocytes andembryos having abnormal metabolic status can be discarded from furtherassisted reproductive methods. The methods provide a way to analyzeembryos/oocytes with a process that is much less invasive than anymethod we are aware of.

The methods for assessing embryo quality are useful for predicting whichembryos have the greatest potential for implantation in order to: (i)increase pregnancy rates with assisted reproductive technologies; (ii)decrease multiple pregnancy rates by justifying transfer of the single“best” embryo; and (iii) appropriately select suitable embryos forcryopreservation and (iv) increase the efficiency of offspring fromtransgenic intervention. The methods for assessing embryo quality arealso useful for assessing the impact or effect of current invasiveprocedures on the oocyte and embryo, including: (i) intracytoplasmicsperm injection; and (ii) blastomere biopsy for pre-implantationcytogenetic diagnosis.

In the current methods we apply FLIM to detection of metabolic state ofembryos and/or oocytes by direct analysis of the embryos or oocytes oranalysis of cells from embryos or analysis of cumulus or granulosa cellssurrounding the oocytes.

As used herein, “oocyte” refers to a female germ cell. The oocyteanalyzed according to the methods of the invention is obtained prior tofertilization, and the analysis is performed in vitro.

“Pre-implantation embryo” or “embryo” is used herein to refer typicallyto an in vitro fertilized oocyte with two pronuclei (up to and includinga blastocyst) but which has not implanted in the lining of the femalereproductive tract. In general, in one embodiment, the pre-implantationembryo contains between about 2 and about 8 cells (i.e., the embryo isassessed between about 18 and about 70 hours post-fertilization),although these ranges may vary among species. Typically, the qualityassessment for a human or a mouse embryo is performed on an embryocontaining between 2 and 8 cells.

“Cell from an embryo” refers to a single cell biopsied from the embryo.Embryo biopsy is a procedure that involves removing one or more cellsfrom the embryo before it is transferred to the mother's uterus. It istypically performed for testing the embryo for specific geneticdisorders. The methods of the present invention can be performed to thecells prior to performing a genetic analysis.

Given the intimate physical and metabolic contact between the cumulusgranulosa cells and the oocyte, FLIM-acquired data of these heterologouscells has been shown to correlate with oocyte health. Such an approachcircumvents any potential harm of illumination to the oocytes, and alsocircumvents any need for clinical trials to prove safety. The terms“cumulus cell” and “granulosa cell” as used herein refer to cumuluscells that are specialized granulosa cells surrounding and nourishingthe oocyte. These cells surround the fully-grown oocyte to form acumulus-oocyte complex (“COC”). The terms cumulus oophorus cells,cumulus granulosa cells, cumulus oophorous granulosa cells,granulosa-cumulus cells are used to make a distinction between thesecells and the other functionally different subpopulation of granulosacells at the wall of the Graafian follicle. Cumulus cells provide keyproducts for the acquisition of developmental competence and differ fromgranulosa cells in their hormonal responses and growth factors theyproduce. The absence of cumulus cells or insufficient numbers of cumuluscells impairs embryo production. Denuded oocytes in culture cannotundergo normal fertilization with standard insemination. Cumulus cellsare required for the successful maturation of oocytes. These cellssynthesize an abundant muco-elastic extracellular matrix, which promotesoocyte extrusion from the follicle, a 20-40 fold increase in the volumeof the cumulus mass, and probably also functions as a selective barrierfor sperm (Salustri, 2000). For cellular and molecular events duringoocyte maturation and the formation of the extracellular matrix of thecumulus-oocyte complex see also: Russel and Salustry (2006) and Kimuraet al (2007). Cumulus cells show high expression of many enzymes of theglycolytic pathway and also neutral amino acid transporters. Theirexpression is promoted by paracrine factors secreted by oocytes (Eppiget al, 2005; Sugiura et al, 2005), which themselves are unable to takeup L-alanine and poorly metabolize glucose for energy production andthus depend on cumulus cells for their provision (Biggers et al, 1967;Colonna and Mangia, 1983; Donahue and Stern, 1968; Eppig et al, 2005;Haghighat and Van Winkle, 1990; Leese and Barton, 1984, 1985). As thecumulus cells provide the growth environment for the oocyte, theirmetabolic state can be used as a proxy of the metabolic state of theoocyte they support. Accordingly, in one aspect of all the embodimentsof the invention the method uses one or more cumulus cells to select anoocyte for in vitro fertilization or for excluding an oocyte from invitro fertilization. The cumulus cells are obtained from around anoocyte prior to insemination or ICSI, and the analysis is performed invitro.

The cells are typically placed in a well suitable for imaging andcomprising cell culture medium at 37° C.

The “reference value” as referred to herein, is typically assessed usingnormal healthy cells of comparable origin, such as normal healthyoocytes, normal healthy embryos, cumulus cells around a normal healthyoocyte or cells from a normal healthy embryo. The reference values aretypically a range from averaged experiments, and are typicallypre-determined although assays including a healthy reference cell ofsimilar origin are also provided.

The methods of the invention use a device that is typically aself-contained, microscope setting, such as a box, such as a table-topmicroscope box that is typically used at in vitro fertilization clinicsto select oocytes for fertilization and embryos for transfer.

The methods are based on use of fluorescence lifetime imaging microscopy(FLIM) of metabolic state of the oocytes or embryos. The interior of thedevice comprises, or consists essentially of the microscope and all theperipherals, as well as an environmental chamber enclosing themicroscope stage. A small slot in the microscope exterior allows custom,multi-well plates containing granulosa cells, oocytes or embryos orcells biopsied from an embryo to be inserted onto the microscope stage.A screen monitor, such as a touch-screen monitor, for example located onthe device's exterior, contains controls and displays acquired data.

The device can be used on oocytes and embryos as well as cumulus cellsand cells isolated from an embryo that can be acquired by any procedureand placed in any media. Thus the methods are easily compatible with thecurrent practices in in vitro fertilization clinics and other settingswhere assessment of embryos or oocytes is performed.

Living cells possess an intricately regulated system of energy-producingand energy-utilizing chemical reactions. Metabolic reactions that areinvolved in energy generation break down macromolecules such ascarbohydrate, lipid, or protein. Most of the energy-generating metabolicpathways of the cell eventually result in the production of acetylcoenzyme A (acetyl CoA). For example, carbohydrates are metabolized topyruvate, which is oxidized to acetyl CoA by the pyruvate dehydrogenasesystem.

Acetyl CoA is completely oxidized in a cyclic series of oxidativereactions alternately referred to as the tricarboxylic acid (TCA) cycle,the Krebs cycle or the citric acid cycle. Although certain of the TCAcycle enzymes are also found in the cytosol, where the enzymes functionin other metabolic pathways, all of the TCA cycle enzymes are located inthe mitochondria. The oxidation of acetyl CoA in one complete TCA cycleresults in the production of two CO₂ molecules, one high energyphosphate bond (such as that present in GTP) and four reducingequivalents, i.e., three NADH and one FADH₂ from three NAD+ and one FAD,respectively.

Measurement of NAHD and FAD has been previously used in cell culturesand analyses of precancerous epithelia (Skala et al., PNAS 104(49):19494-19499, 2007). Skala et al. showed that a decrease in protein-boundNADH, and an increase in protein bound NAD fluorescence lifetime wasassociated with different states of epithelial cancer. It has also beenshown that the fluorescent lifetime of free and protein-bound NADHdecrease with hypoxia (Bird et al. Cancer Res. 65: 8766-8773, 2005;Schneckenburger et al., J. Fluorescence 14: 649-654, 2004) and decreasein pre-cancers of hamster cheek pouch model of oral cancer in vivo(Skala et al. J. Biomed. Opt. 12:024014, 2007).

It has been suggested that measuring pyruvate levels in oocyte/embryoculture media as a means of assessing metabolic activity is inadequatefor a number of reasons. For example, the contribution of pyruvate tothe media from cumulus cells surrounding the oocyte cannot be predicted.In addition, the metabolic profile of the oocyte/embryo is complicatedby the changing nutrient (e.g., substrate) requirements of thedeveloping embryo from immature oocyte to blastocyst. In the oocyte andearly stage embryo, the vast majority of pyruvate that is taken up bythe cell is channeled via acetyl CoA into the mitochondrial TCA cycleand oxidative phosphorylation (Wales, R. et al, (1970) Aust. J. Biol.Sci. 23, 877-887). Pyruvate is required to support the first and secondcleavage divisions of the embryo in culture (Biggers, J. et al, (1967)Proc. Natl. Acad. Sci. U.S.A. 58(2), 560-567), whereas glucose is unableto support development until the four-cell stage. Glucose is thepredominant nutrient required by the blastocyst (Brinster, R. et al,(1966) Exp. Cell Res. 42, 303-315). Cells in culture exhibit differentnutrient requirements over time. For example, the oxidative metabolismof cultured cells declines over time; such cells become increasinglydependent on anaerobic glycolysis with concomitant lactate production(Morgan, M. et al, (1981) Biosci. Rep. 1, 669-686). Thus, for example,mouse blastocysts which have been cultured in vitro produce almost twiceas much lactate as blastocysts which are freshly collected (Gardner, D.et al, (1990) J. Reprod. Feral. 88, 361-368). Therefore, the simplemeasurement of pyruvate (nutrient) in culture media is not necessarily areliable or accurate measure of the mitochondrial or metabolic status ofthe oocyte/embryo.

The oocyte/embryo contains a steady-state concentration of NADH that ispresent in the cell(s) as a result of metabolic reactions taking placein the mitochondria (e.g., tricarboxylic acid cycle and electrontransport) and in the cytosol (e.g., glycolysis). Although one mightexpect to find a correlation between metabolic activity andoocyte/embryo viability, past efforts failed to establish any suchdirect correlation. For example, a study by Conaghan et al, J. Assist.Reprod. Genet 10(1): 21-30, 1993, reported that pyruvate uptake by humanembryos was not predictive of those that successfully implanted.

The findings in U.S. Pat. No. 5,541,081 suggested that to obtaininformation about the metabolic state of an embryo/oocytes using NADH,one must first reduce the endogenous NADH concentration of theoocyte/embryo by placing it in a control medium and obtaining at leastone control NAHD fluorescence measurement. After the controlmeasurement, the oocyte/embryo was then subjected to a different mediumwith a nutrient for a time period and the change in the NADHconcentration was observed. This analysis is not only time consuming butalso subjects the embryo/oocyte to an additional and unnecessary stresswhen it is moved from special medium to another.

Contrary to the prior reported methods for embryo assessment, the methodof the present invention allows direct analysis of NADH and/or FADinside an embryo/oocyte/cumulus cell/cell from embryo without subjectingthe embryo/oocyte/cumulus cell/cell from embryo to changes in itsculture medium. Due to the specificity of FLIM, we have shown that wecan observe the fluorescent lifetime of protein bound and free NADHand/or FAD which we have found is indicative of the metabolic activityof the embryo/oocyte and predictive of, e.g., implantation success.

Fluorescence-lifetime imaging microscopy (FLIM) is an imaging techniquefor producing an image based on the differences in the exponential decayrate of the fluorescence from a fluorescent sample. FLIM can be used asan imaging technique in confocal microscopy, two-photon excitationmicroscopy, and multiphoton tomography.

The lifetime of the fluorophore signal, rather than its intensity, isused to create the image in FLIM. This has the advantage of minimizingthe effect of photon scattering in thick layers of sample.

A fluorophore which is excited by a photon will drop to the ground statewith a certain probability based on the decay rates through a number ofdifferent (radiative and/or nonradiative) decay pathways. To observefluorescence, one of these pathways must be by spontaneous emission of aphoton. This can be utilized for making non-intensity based measurementsin chemical sensing.

Fluorescence lifetimes can be determined in the time domain by using apulsed source.

Time-correlated single-photon counting (TCSPC) is usually employed. Morespecifically, TCSPC records times at which individual photons aredetected by something like a photo-multiplier tube (PMT) or an avalanchephoto diode (APD) after a single pulse. The recordings are repeated foradditional pulses, and after enough recorded events one is able to builda histogram of the number of events across all of these recorded timepoints. This histogram can then be fit to a function that containsparameters of interest, and thus the parameters can be accordingly beextracted. 16˜64 multichannel PMT systems have been commerciallyavailable, whereas the recently demonstrated CMOS single-photonavalanche diode (SPAD)-TCSPC FLIM systems can offer additional low-costoptions.

Pulse excitation is still used in the gating method. Before the pulsereaches the sample, some of the light is reflected by a dichroic mirrorand gets detected by a photodiode that activates a delay generatorcontrolling a gated optical intensifier (GOI) that sits in front of yourCCD detector. The GOI only allows for detection for the fraction of timewhen it is open after the delay. Thus, with an adjustable delaygenerator, one is able to collect fluorescence emission after multipledelay times encompassing the time range of the fluorescence decay of thesample.

Alternatively, fluorescence lifetimes can be determined in the frequencydomain by a phase-modulated method. The intensity of a continuous wavesource is modulated at high frequency, by an acousto-optic modulator forexample, which will modulate the fluorescence. Since the excited statehas a lifetime, the fluorescence will be delayed with respect to theexcitation signal, and the lifetime can be determined from the phaseshift. Also, y-components to the excitation and fluorescence sine waveswill be modulated, and lifetime can be determined from the modulationratio of these y-components. Hence, 2 values for the lifetime can bedetermined from the phase-modulation method. Consequently, if thelifetimes that are extracted from the y-component and the phase do notmatch, it means that you have more than one lifetime species in yoursample.

FLIM has primarily been used in biology as a method to detectphotosensitizers in cells and tumors as well as FRET in instances whereratiometric imaging is difficult. The technique was developed in thelate 1980s and early 1990s (Bugiel et al. 1989. König 1989) before beingmore widely applied in the late 1990s (Oida T, Sako Y, Kusumi A (March1993). “Fluorescence lifetime imaging microscopy (flimscopy).Methodology development and application to studies of endosome fusion insingle cells”. Biophys. J. 64 (3): 676-85). In cell culture, it has beenused to study EGF receptor signaling (Wouters F S, Bastiaens P I(October 1999). “Fluorescence lifetime imaging of receptor tyrosinekinase activity in cells”. Curr. Biol. 9 (19): 1127-30) and ErbB1receptor trafficking (Verveer P J, Wouters F S, Reynolds A R, BastiaensP I (November 2000). “Quantitative imaging of lateral ErbB1 receptorsignal propagation in the plasma membrane”. Science 290 (5496):1567-70). FLIM imaging is particularly useful in neurons, where lightscattering by brain tissue is problematic for ratiometric imaging(Yasuda R (October 2006). “Imaging spatiotemporal dynamics of neuronalsignaling using fluorescence resonance energy transfer and fluorescencelifetime imaging microscopy”. Curr. Opin. Neurobiol. 16 (5): 551-61). Inneurons, FLIM imaging using pulsed illumination has been used to studyRas (Harvey C D, Yasuda R, Zhong H, Svoboda K (July 2008). “The spreadof ras activity triggered by activation of a single dendritic spine”.Science 321 (5885): 136-40), CaMKII, Rac, and Ran (The design ofForester (fluorescence) resonance energy transfer (FRET)-based molecularsensors for Ran GTPase, in press P. Kalab, J. Soderholm, Methods (2010)family proteins). FLIM has also been used in clinical multiphotontomography to detect intradermal cancer cells as well as pharmaceuticaland cosmetical compounds.

The cells in the present method can be analyzed in any suitable cellculture medium used for embryo/oocyte/cumulus cell/cell from embryo.Thus, the present methods avoid subjecting the cells to anyextraordinary medium changes. Moreover, because there is no need tochange the metabolic state of the cell, like e.g., in the '081 patent,no additional time is needed for the analysis, making the analysis fastand convenient.

The measurement consists of acquiring a single FLIM image per cell,which can be obtained rapidly and non-invasively or minimallyinvasively. Typically, it takes about 1-5 minutes, sometimes 30 secondsto 2 minutes to load a sample containing the cells to be studied andonly seconds to acquire the data after which the oocyte/embryo that hasbeen analyzed can either be selected for further fertilization orimplantation or discarded as not optimally fit for these procedures.

The FLIM measurement is taken directly inside the embryo using theautoflourescence of nicotinamide adenine dinucleotide (NADH) or flavinadenine dinucleotide (FAD) both molecules involved in cellularmetabolism.

In the methods of the present invention, the acquired data are typicallysubsequently averaged over the entire cell, because subcellularinformation is unnecessary, producing one FLIM curve per cell.

While we have used mouse embryos and oocytes in our examples providedherein, the assay will not need to be altered when analyzing humancells. The metabolic state of human and mouse embryos and oocytes arecomparable during these stages of development and thus the resultsobtained with mouse oocytes/embryos can be directly applied to humancells as well.

In essence, the method of the invention comprises exposing a test cellto a fluorescence lifetime imaging microscope (FLIM) to acquire afluorescence lifetime histogram of auto-fluorescence of endogenous NADHand/or FAD for the test cell. The test cell can be an oocyte or anoocyte-associated cumulus cell or an embryo or a cell from the embryo.

Before the exposure, the test cell can be in or be placed in any normalcell culture medium used in maintaining the embryos/oocytes at theclinic. Culture media for embryo development should meet the metabolicneeds of pre-implantation embryos by addressing amino acid and energyrequirements based on the specific developmental stage of the embryo.

Various culture media are and have been used in ART methods and any ofthem can be used in the methods of the invention. The following providessome examples of culture media. Culture media, like Earle, Ham's F10,Tyrode's T6 and Whitten's WM1 were based on different salts and wereconstructed to support the development of somatic cells and cell linesin culture. These culture media, known as physiological salt solutionswere used by Robert Edwards for his first successful In VitroFertilization (IVF). These media were formulated for use with or withoutserum supplementation, depending on the cell type being cultured. TheHam's Nutrient Mixtures were originally developed to support growth ofseveral clones of Chinese hamster ovary (CHO) cells, as well as clonesof HeLa and mouse L-cells.

Menezo et al., 1984, suggested adding serum albumin as a source foramino acids. The serum protein ensures that oocytes and embryos do notadhere to the glass surface of the pipette used to manipulate them. Themedium entitled B2, is still in use today. In 1985 Quinn et al.published in the journal Fertility and Sterility a formula entitledHuman Tubal Fluid (HTF), which mimics the in vivo environment to whichthe embryo is exposed. The formulation of HTF was based on the knownchemical composition of the fluids in human fallopian tubes as known atthat time. This medium is based on a simple balanced salt solutionwithout amino acids; however, the concentration of potassium wasadjusted to that measured in the human female reproductive tract. Thismedium was found to be better compared with earlier media developed.

The supplementation of the HTF medium with either whole serum or withserum albumin became a gold standard for the production of culturemedium for human embryos transferred on day 2 or day 3 of culture, forexample 1-4 days in culture, 2-3 days in culture, 2-4 days in culture.

It is typically considered that culturing embryos involves addressingspecific needs depending on the developmental stage of the embryo.Energy source requirements evolve from a pyruvate-lactate preferencewhile the embryos, up to the 8-cell stage, are under maternal geneticcontrol, to a glucose based metabolism after activation of the embryonicgenome that supports their development from 8-cells to blastocysts. Thisobservation lead to the development of the first commercial media. Theculture media developed was based on HTF: both media were free ofinorganic phosphate, glucose and amino acids. Pool and his colleaguesformulated HTF which was free of glucose and phosphate. Cleaving embryosuse pyrovate and lactate as energy sources and non-essential amino acids(NEAA) for protein metabolism. From the 8-cell stage the major energysource is glucose and for protein metabolism the embryos use essentialamino acids (EAA). These findings lead to development of composition oftwo culture media G1 and G2 that are to be used in sequence. G1 supportsthe in-vitro development of the fertilized oocyte, the zygote, to the8-cell stage, and G2 from 8-cells to blastocyst. Several modificationsto these media also exist and are well known to one skilled in the art.Sequential media are now being used successfully in IVF treatment allover the world.

Any of the above-discussed media may be used when imaging the test cellsaccording to the methods of the invention.

The typical composition of the embryo culture medium includes: culturemedia containing a phosphate buffer or Hepes organic buffer are used forprocedures that involve handling of gametes outside of the incubator,flushing of follicles and micromanipulation. The pH and osmolality formost culture media utilize a bicarbonate/CO₂ buffer system to keep pH inthe range of 7.2-7.4. The osmolarity of the culture medium should be inthe range of 275-290 mosmol/kg. Similar conditions should optimally bemaintained while imaging the cells according to the methods of theinvention.

In addition, the human oocyte is temperature-sensitive and a humidifiedincubator with a temperature setting of 37.0-37.5° C. should be used foroocyte fertilization and embryo culture. Similar temperature shouldoptimally be maintained while imaging the cells according to the methodsof the invention.

Embryos should be cultured under paraffin oil, which preventsevaporation of the medium preserving a constant osmolarity. The oil alsominimizes fluctuations of pH and temperature when embryos are taken outof the incubator for microscopic assessment. Paraffin oil can be toxicto gametes and embryos; therefore, batches of oil must be screened andtested on mouse embryos before use in culture of human embryos. The oildoes not need to be removed to perform the FLIM analysis of theinvention.

The medium is also composed of 99% water. Purity of the water isimportant, and is typically achieved by ultrafiltration.

Albumin or synthetic serum are typically added in concentrations of 5 to20% (w/v or v/v, respectively). The commercial media typically includessynthetic serum in which the composition is well known.

Commercial IVF media typically comprises, for example, one or more ofthe following components: synthetic serum, recombinant albumin, saltsolution in MTF, NaCl, KCl, KH₂PO₄, CaCl₂2H₂O, MgSO₄7H2O, NaHCO₃, andcarbohydrates.

Carbohydrates are present in the female reproductive tract. Theirconcentrations vary throughout the length of the oviduct and in theuterus, and are also dependent on the time of the cycle.

Together with the amino acids they carbohydrates are the main energysource for the embryo. Culture media that support the development ofzygotes up to 8-cells contain pyruvate and lactate. Some commercialmedia are glucose free, while others add a very low concentration ofglucose to supply the needs of the sperm during conventionalinsemination.

Media that support the development of 8-cell embryos up to theblastocyst stage contain pyruvate and lactate in low concentrations anda higher concentration of glucose.

Amino acids supplement of the culture medium with amino acids isnecessary for embryo development. Media that support the development ofzygotes up to 8-cells are often further supplemented with non-essentialamino acids. Proline, serine, alanine, aspargine, aspartate, glycine,glutamate. Media that support the development of 8-cell embryos up tothe blastocyst stage are typically supplemented with essential aminoacids: Cystine, histadine, isolucine, leucine, lysine, methionine,valine, argentine, glutamine, phenylalanine, therionine, tryptophane.

The majority of ART laboratories use culture media containingantibiotics to minimize the risks of microbial growth. The most commonlyused antibiotics being Penicillin (β-lactam Gram-positive bacteriadisturbs cell wall integrity) and Streptomycin (AminoglycosideGram-negative bacteria disturbs protein synthesis). The anti-bacterialeffect of penicillin is attributed to its ability to inhibit thesynthesis of peptidoglycan, unique glycoproteins of the bacterial cellwall. Streptomycin and gentamycin belong to the aminoglycoside group ofantibiotics which exert their antibacterial effect by inhibitingbacterial protein synthesis. The use of genthamicine is stillcontroversial and it is not being used by every laboratory.

Often, the culture medium also comprises EDTA which is used as achelator in medium that supports the embryo from the zygote stage to8-cells and prevents abnormal glycolysis.

As noted before, the methods of the invention are not dependent on thetype of the medium. The cells should remain in the medium and conditionsthey are cultured to avoid additional stress to them during the FLIManalysis.

The method further comprises averaging the fluorescence lifetimehistogram of NADH auto-fluorescence or FAD auto-fluorescence or bothover the entire test cell, or the cytoplasm the test cell, ormitochondria of the test cell.

To be able to select the healthy cells for further assisted reproductivemethods, the method also comprises comparing the averaged fluorescencelifetime histogram from the test cell to an averaged fluorescencelifetime histogram reference value to determine if the measured averagedfluorescence lifetime histogram from the test cell differs statisticallyfrom that of the reference value. The comparing is typically made usinga non-human machine typically using a computer executable software whichincludes a comparison between a reference value and the value from eachindividual cell FLIM analyses.

Similarly to NADH, also autofluorescence of FAD can be analyzed usingFLIM in the methods of the present invention.

The FLIM curves of NADH crom cells exhibit a double exponential decaywith a long lifetime (about 2.5 nanoseconds (ns)) corresponding toprotein bound NADH and a short lifetime (about 0.4 ns) corresponding tofree NADH. Thus, the FLIM curve is a double exponential with therelative fraction of the long and short lifetimes reflecting therelative fraction of protein bound and free NADH. This provides a directreadout of the metabolic state of the cell (Lacowciz et al., 1992). Thelong lifetime might vary from 1-3 (nanoseconds) ns and the short lifetime might vary from 0.2-0.7 ns.

The precise value of these lifetimes in the free and bound statesdepends on a variety of cellular factors, such as pH (Ogikubo et al.,2011). The relative fraction of the long and short lifetimes, and theprecise value of these lifetimes is typically determined by using aleast-squares fit. However, Baysian inferences approaches, may alloweven more precise parameter estimates and reference estimates with fewernumber of photons, allowing even less light to be used to obtain furtherreliability and further minimizing sample exposure.

The absorption and fluorescence spectra of NADH (the reduced form) havebeen well characterized at different levels of organization, i.e., insolution, mitochondria and cell suspensions, tissue slices, and organsin vitro and in vivo. NADH has an optical absorption band at about 300to 380 nm and a fluorescence emission band at 420 to 480 nm. The spectraare considered the same, although there are small differences in theshape and maxima of the spectra for different environments andmeasurement conditions. However, there is a universal agreement that theintensity of the fluorescence band, independent of the organizationlevel of the environment, is proportional to the concentration ofmitochondrial NADH (the reduced form), particularly when measured invivo from a tissue (see, e.g., review by Avraham Mayevsky and Gennady G.Rogatsky, Am J Physiol Cell Physiol February 2007 vol. 292 no. 2C615-C640).

Accordingly, in some aspects of all the embodiments of the invention,the direct autofluorescence of NADH can be analyzed with FLIM using awavelength of about 740 nm in two-photon fluorescence excitation andusing an emission bandpass filtered centered around about 460 nm.

In some aspects of all the embodiments of the invention, the directautofluorescence of NADH can also be analyzed with FLIM using awavelength of about 340 nm in one-photon fluorescence excitation andusing an emission bandpass filtered centered around about 460 nm.

In some aspects of all the embodiments of the invention, the directautofluosescence of FAD can be analyzed with FLIM using a wavelength ofabout 900 nm in two-photon fluorescence excitation and using an emissionbandpass filtered centered around about 550 nm.

The direct autofluorescence of FAD can be analyzed with FLIM using awavelength of about 450 nm in one-photon fluorescence excitation andusing an emission bandpass filtered centered around about 550 nm.

The embryo analyzed according to the methods of the invention is apre-implantation embryo, and the analysis is performed in vitro.Similarly, a cell obtained from an embryo is obtained from apre-implantation embryo and the cell biopsy is performed in vitro.

One can combine the analysis using FLIM of NAHD and FAD by simplyexposing the cells sequentially to wavelength suitable for NADH and thento FAD or wavelength suitable for imaging the fluorescence lifetime ofFAD and then NAHD. Accordingly, in some aspects of all the embodimentsof the invention the method comprises a sequential analysis of FAD andNADH.

The analysis typically only takes a short time, such as 30 seconds to 5minutes and can be multiplexed and automated.

Thus, we provide a method for assessing the quality of an oocyte or anembryo, the method comprising (a) exposing, in a medium, granulosacell(s), oocyte, the embryo or cell(s) from the embryo to a fluorescencelifetime imaging microscopy (FLIM) to acquire a fluorescence lifetimehistogram of auto-fluorescence of NADH in the granulosa cells, oocyte,the embryo or cell(s) from the embryo; (b) averaging the fluorescencelifetime histogram of auto-fluorescence of NADH over the entiregranulosa cell, oocyte, the embryo or cell(s) from the embryo; (c)fitting the averaged fluorescence lifetime histogram ofauto-fluorescence of NADH to a sum of two exponentials; and (d)selecting for in vitro fertilization or implantation the oocyte orembryo when one detects a cell, whether granulosa cells, oocyte, theembryo or cell(s) from the embryo, in which the alpha is less than analpha reference value and the beta is less than a beta reference valueor discarding the oocyte or embryo from in vitro fertilization when onedetects a cell, whether granulosa cells, oocyte, the embryo or cell(s)from the embryo, in which the alpha is equal or more than the alphareference value and if the beta is equal or more than the beta referencevalue. The cell can be an embryo, oocyte, or a cell extracted from enembryo or a cumulus cell surrounding an oocyte. In some aspects, themethod further comprises a step of in vitro fertilizing the oocyte whenone detects a cell, either oocyte or one or more cumulus cell fromaround the oocyte with the alpha is less than an alpha reference valueand the beta is less than a beta reference value, or implanting theembryo when one detects a cell from the embryo or an embryo with thealpha is less than an alpha reference value and the beta is less than abeta reference value.

In some aspects of all the embodiments of the invention, the alphareference value is between 1 and 4, such as 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, or 2-3, or 2-4,or 1-2, or 1-3. In some aspects of all the embodiments of the invention,the alpha reference value is 1.7.

In some aspects of all the embodiments of the invention, the betareference value is 2000 ps-3000 ps, or 2000-2500, or 2500-3000, or2000-2250, or 2000-2750. In some aspects of all the embodiments the betareference value is 2250 ps (picoseconds). FIG. 1 provides an example ofsuitable alpha and beta values for evaluation of oocytes.

While our preliminary analyses have been performed using mouse oocytesas a model. The metabolic state of oocytes and embryos in both humansand mice is practically identical. Therefore, the reference valuesobtained from the metabolic state FLIM analysis in a mouse cumulus cell,oocyte and/or embryo, can be directly applied to human oocyte and embryoanalysis. The reference values can also be obtained from human cumuluscell, oocyte and/or embryo.

In some aspects of all the embodiments of the invention, the FLIM ofNADH is performed using a wavelength of about 740 nm in two-photonexcitation and using an emission bandpass filter centered around about460 nm.

In some aspects of all the embodiments of the invention, one obtainsFLIM images at different times instead of only acquiring a single image.This kind of time-lapse data would be richer, and thus can provide moreinformation about the cells. However, it will require cells to be in thedevice for longer periods of time and they would be exposed to morelight, thereby slightly increasing the invasiveness of the method.

The described method is typically performed using a system that containsnearly all of the components of a traditional microscope. Thus themethod can be combined with analysis of morphological time-lapse data aswell.

There are three possible options for performing the FLIM measurements,all of which can be used in the methods of the present invention:two-photon excitation with a point detector, one-photon excitation witha point detector, or one-photon detection with an area detector (i.e. acamera). The table below indicates the advantages of each of theoptions. We used the two-photon method in our examples.

Two-Photon One-Photon One-Photon Point Detector Point Detector CameraSensitivity Highest Medium Lowest Cost Highest Medium Lowest RobustnessLowest Robustness Medium Robustness Most Robust Complexity SimpleOptical Setup with Complex Optical Setup Simple Optical Setup StageScanning Imaging with Stage Scanning Imaging Safety Long WavelengthShorter Wavelength Shorter Wavelength Considerations (reduces possibledamage)

Examples

We obtained preliminary data on FLIM of NADH in mouse oocytes. Thepreliminary data was acquired on a FLIM system. The microscope consistsof a ti-sapphire femtosecond laser (Spectra-Physics), an invertedmicroscope base (Nikon), a scan head (Becker & Hickl), a hybrid PMTdetector (Hamamatsu), and electronics for time correlated single photoncounting (Becker & Hickl). This microscope was assembled to acquire thepreliminary data.

Oocytes were placed in a medium on the microscope stage and imaged. Asingle image of each oocyte was analyzed by averaging the FLIM data overthe entire oocyte. The acquired fluorescence lifetime histogram fromNADH, averaged over the entire oocyte, was fit to a sum of twoexponentials. The parameter alpha, is the ratio of the amplitude of thetwo exponentials, the parameter, beta, is the lifetime of the longerexponential (in picoseconds).

Unperturbed oocytes exhibit a range of values of alpha and beta (FIG. 1,circles). As seen in FIG. 1, oocyte quality depends on oocyte metabolicstate. Oocyte metabolic state can be rapidly, non-invasively, andquantitatively measured by Fluorescence Lifetime Imaging Microscopy(FLIM) of NAD or NADH. Two parameters (alpha and beta) are extractedfrom FLIM measurements of NADH (or NAD) in oocytes. In the example, weused mouse oocytes but similar calculations are expected to work forhuman oocytes, as the mammalian cells, such as mouse and human cells arerelatively similar, particularly relating to their metabolic state, atthis stage. Each point corresponds to data from a single oocyte.Perturbing oocytes by specific metabolic inhibitors (black crosses), ornon-specific damage (triangles) causes both parameters to increase.Unperturbed oocytes (circles) exhibit a range of values of alpha andbeta. These parameters are indicative of oocyte quality.

Alpha and beta both increase when oocytes are perturbed by eitherapplying a metabolic inhibitor (FIG. 1, black circles) or greatlyincreasing laser power to intentionally damage the oocytes (FIG. 1,triangles). Such damage does not occur at the low laser powers used forimaging.

Our results show that low values of alpha and beta are indicative ofoocyte and embryo health. All perturbed oocytes are found in the upperquadrant with alpha greater than 1.7 and beta greater than 2250. Thisshows that these values of alpha and beta can be used as cutoffs withoocytes above these values rejected as likely being unhealthy, andoocytes below these values deemed healthy and selected for use.

The metabolism of human and mouse oocytes are very similar, and thus,cutoff values of alpha and beta established for mice can be used forhuman analyses as well.

The values of alpha and beta are obtained quantitatively, rapidly, andobjectively. Thus the described approach is highly useful in in vitrofertilization clinics.

REFERENCES

The references cited herein and throughout the specification are hereinincorporated by reference in their entirety.

-   Botros, L., Sakkas, D. and Seli, E. Metabolomics and its application    for non-invasive embryo assessment in IVF. Molecular Human    Reproduction Vol. 14, No. 12 pp. 679-690, 2008.-   Lakowciz, J. R., Szmacinski, H., Nowaczyk, K., and Johnson, M. L.    Fluorescence lifetime imaging of free and protein-bound NADH.    PNAS. 1992. 89. 1271-1275-   Ogikubo, S., et al., Intracellular pH Sensing Using Autofluorescence    Lifetime Microscopy. Journal of Physical Chemistry B. 2011. 115,    10385-10390.-   Scott R T, Kerry F, Su J, Tao X, Scott K, Treff N. Comprehensive    chromosome screening is highly predictive of the reproductive    potential of human embryos: a prospective, blinded, non-selection    study. Fertil Steril 2012; 97:870-5.-   Vergouw C G, Kieslinger D C, Kostelijk E H, Botros L L, Schats R,    Hompes P G, Sakkas D, Lambalk C B. Day 3 embryo selection by    metabolomic profiling of culture medium with near-infrared    spectroscopy as an adjunct to morphology: a randomized controlled    trial. Hum Reprod. 2012; 27(8):2304-11.-   Seli, E. and Brommer, J. G. Assessment of embryo viability in    assisted reproductive technology: shortcomings of current approaches    and the emerging role of metabolomics. Current Opinion in Obstetrics    and Gynecology. 2008, 20:234-241-   Society for Assisted Reproductive Technology (SART). Assisted    reproductive technology success rates. National summary and    fertility clinic reports. Atlanta, Ga.: Centers for Disease Control    and Prevention; 2005-   Stringaria, C., Cinquinb, A., Cinquinb, O., Digmana, M. A.,    Donovan, P. J., and Grattona, E. Phasor approach to fluorescence    lifetime microscopy distinguishes different metabolic states of germ    cells in alive tissue. PNAS. 2011. 108: 33. 13582-13587-   Uyar A, Seli E. Embryo assessment strategies and their validation    for clinical use: a critical analysis of methodology. Curr Opin    Obstet Gynecol. 2012 June; 24(3):141-50-   Zipfel W R, et al. (2003) Live tissue intrinsic emission microscopy    using multiphotonexcited native fluorescence and second harmonic    generation. Proc Natl Acad Sci USA 100:7075-7080. Botros, L.,    Sakkas, D. and Seli, E. Metabolomics and its application for    non-invasive embryo assessment in IVF. Molecular Human Reproduction    Vol. 14, No. 12 pp. 679-690, 2008-   Lakowciz, J. R., Szmacinski, H., Nowaczyk, K., and Johnson, M. L.    Fluorescence lifetime imaging of free and protein-bound NADH.    PNAS. 1992. 89. 1271-1275-   Ogikubo, S., et al., Intracellular pH Sensing Using Autofluorescence    Lifetime Microscopy. Journal of Physical Chemistry B. 2011. 115,    10385-10390.-   Scott R T, Kerry F, Su J, Tao X, Scott K, Treff N. Comprehensive    chromosome screening is highly predictive of the reproductive    potential of human embryos: a prospective, blinded, non-selection    study. Fertil Steril 2012; 97:870-5.-   Vergouw C G, Kieslinger D C, Kostelijk E H, Botros L L, Schats R,    Hompes P G, Sakkas D, Lambalk C B. Day 3 embryo selection by    metabolomic profiling of culture medium with near-infrared    spectroscopy as an adjunct to morphology: a randomized controlled    trial. Hum Reprod. 2012; 27(8):2304-11.-   Seli, E. and Brommer, J. G. Assessment of embryo viability in    assisted reproductive technology: shortcomings of current approaches    and the emerging role of metabolomics. Current Opinion in Obstetrics    and Gynecology. 2008, 20:234-241-   Society for Assisted Reproductive Technology (SART). Assisted    reproductive technology success rates. National summary and    fertility clinic reports. Atlanta, Ga.: Centers for Disease Control    and Prevention; 2005-   Stringaria, C., Cinquinb, A., Cinquinb, O., Digmana, M. A.,    Donovan, P. J., and Grattona, E. Phasor approach to fluorescence    lifetime microscopy distinguishes different metabolic states of germ    cells in alive tissue. PNAS. 2011. 108: 33. 13582-13587-   Uyar A, Seli E. Embryo assessment strategies and their validation    for clinical use: a critical analysis of methodology. Curr Opin    Obstet Gynecol. 2012 June; 24(3):141-50-   Zipfel W R, et al. (2003) Live tissue intrinsic emission microscopy    using multiphotonexcited native fluorescence and second harmonic    generation. Proc Natl Acad Sci USA 100:7075-7080.

1. A method for assessing the quality of an oocyte or an embryo, themethod comprising (a) exposing a test cell selected from the oocyte oran oocyte-associated cumulus cell or the embryo or a cell from theembryo to a fluorescence lifetime imaging microscope (FLIM) to acquire afluorescence lifetime histogram of auto-fluorescence of endogenous NADHor FAD for the test cell; (b) averaging the fluorescence lifetimehistogram of NADH auto-fluorescence or FAD auto-fluorescence or bothover the entire test cell, or the cytoplasm the test cell, ormitochondria of the test cell; (c) comparing the averaged fluorescencelifetime histogram from the test cell to an averaged fluorescencelifetime histogram reference value to determine if the measured averagedfluorescence lifetime histogram from the test cell differs statisticallyfrom that of the reference value; and (d) if the averaged fluorescencelifetime histogram from the test cell does not differ statistically fromthe reference value then selecting the oocyte for in vitro fertilizationor embryo for implantation and if the averaged fluorescence lifetimehistogram from the test cell differs statistically from the referencevalue then excluding the oocyte from in vitro fertilization or embryofrom implantation.
 2. The method of claim 1 comprising the step ofestablishing the statistical significance by fitting lifetime histogramsto a sum of two exponentials and the parameters of the measured cellsare deemed to fall within or not fall within the range of parametersfound in the healthy cells.
 3. The method of claim 1, wherein theparameters to be compared between the measurements and the healthy cellsare alpha, defined as the ratio of the amplitude of the twoexponentials, and beta, defined as the lifetime of the longerexponential.
 4. The method of claim 1, wherein the maximum value ofalpha from the healthy cells is a specific value within the range1.0-4.0 and the corresponding maximum value of beta of healthy cells isin the range 2000 ps-3000 ps.
 5. The method of claim 1, wherein the FLIMis performed using a wavelength of about 740 nm in two-photonfluorescence excitation and using an emission bandpass filtered centeredaround about 460 nm.
 6. The method of claim 1, wherein the FLIM isperformed using a wavelength of about 340 nm in one-photon fluorescenceexcitation and using an emission bandpass filtered centered around about460 nm.
 7. The method of claim 1, wherein the FLIM is performed in thetime domain.
 8. The method of claim 1, wherein the FLIM is performed inthe frequency domain.
 9. The method of claim 1, wherein the FLIM isperformed using a wavelength of about 900 nm in two-photon fluorescenceexcitation and using an emission bandpass filtered centered around about550 nm.
 10. The method of claim 1, wherein the FLIM is performed using awavelength of about 450 nm in one-photon fluorescence excitation andusing an emission bandpass filtered centered around about 550 nm.